The development of gene transfer methods into liver is very attractive because the liver is the site of production for many secreted proteins (e.g., blood clotting factors VIII and IX) as well as the site of many inherited and acquired disorders including hemophilia, hypercholester-olemia, and hepatitis. The large size of this organ and its regenerative capabilities allow for transient damage and may be exploited to enhance gene transfer. Furthermore, the recently developed technique of intravascular delivery of naked plasmid DNA expression vectors allows high efficiency transfection of hepatocytes [Zhang et al. Hum Gene Ther. 1999 10(10):1735-1737]. Despite this great promise, the challenge is to both efficiently transfer and stably express transgenes in liver.
The injection of naked plasmid DNA (pDNA) into liver or tail vein vessels leads to high levels of foreign gene expression in rodent hepatocytes. Zhang et al. [Zhang et al. Hum Gene Ther. 1999 10(10):1735-1737] observed almost milligram quantities of foreign protein could be produced from >5% of the hepatocytes one day after injection. Such levels of gene transfer are sufficient to treat several common genetic diseases. Preliminary experiments have shown that intravascular injection in mice of an expression vector in which the human factor VIII gene is under transcriptional control of the cytomegalovirus promoter resulted in plasma levels of ˜500 ng/ml after one day. This level is equivalent to 100-250% of the normal human plasma levels for this protein. For hemophilia A and B, their clinical courses are greatly influenced by the percentage of normal serum levels of factor VIII or IX: <2%, severe; 2-5%, moderate; and 5-30% mild [Kay and High K. Proc Natl Acad Sci USA. 1999 96(18):9973-9975]. A gene therapy that provides these factors continuously, even at 2-5% normal levels, would tremendously improve the patients' quality of life. Therefore, if expression levels were stable, the efficiency of the current naked DNA in vivo transfection method would be sufficient for human gene therapy of hemophilia A as well as several other genetic diseases including: hemophilia B, phenylketonuria, hypercholesterolemia, urea cycle disorders and organic acidurias. Expression of these factors would ideally be sustained for the lifetime of the patient, or at least for months or years.
Unstable expression of foreign genes in the liver has been observed using both viral and non-viral approaches [Yew et al. Mol Ther. 2001 4(1):75-82; Herweijer et al. J Gene Med. 2001 3(3):280-291]. Using a variety of techniques for delivery of DNA to a cell, foreign gene expression typically falls rapidly after 1-2 days. Promoter inactivation appears be the primary cause of this initial phase of expression loss [VandenDriessche et al. Curr Gene Ther 2001 September; 1(3):301-315]. The use of viral promoters, such as CMV, RSV, and SV40 promoters, while resulting in higher expression levels than cellular promoters in in vitro experiments, were considerably less effective in vivo. Furthermore, despite high levels of expression in vivo soon after gene transfer, expression diminished over time due to promoter shut off. Additionally, several studies have found that in vivo expression from a variety of promoters in the liver following adenoviral delivery quickly decreased within the first few days. For adenoviral delivery, unstable expression may have been partly caused by immune effects against the vector particle or viral gene products. Immune response to foreign gene products may also be partially responsible for the decreased expression of transgenes in vivo.
To overcome the shutoff of viral promoters, much work has gone into identification of promoter and enhancer sequences that might provide long term expression. Viral promoters generally expressed at very high levels one day after injection, but expression fell precipitously after day one. Conversely, tissue specific promoters, such as the liver-specific albumin promoter, often gave the desired sustained expression, but only at expression levels below therapeutic amounts [Herweijer et al. J Gene Med. 2001 3(3):280-291]. Hybrid promoters containing both viral and mammalian sequences have also been constructed in attempts to enable stable, high expression in vivo. For example, elements of the hepatitis B virus (HBV) enhancer in conjunction with the CMV promoter resulted in more sustained expression in hepatocyte cell cultures.
In eukaryotic cells, transcription of RNA polymerase II transcribed genes starts with the assembly of a preinitiation complex on the promoter of a gene. The promoter typically consists of a TATA box, CAAT sequences, and other binding motifs, called cis regulatory elements, to which transcription factors bind. The formation of this preinitiation complex is enhanced or impeded by other factors binding to the DNA. The binding of these positive and negative transcriptional regulators to the promoter and enhancer regions of a gene influences recruitment of the RNA polymerase preinitiation complex to the gene, the probability of initiation of transcription by the bound RNA polymerase, and the efficiency of the RNA polymerase in transcribing the gene. Binding of a transcriptional activator to its cognate regulatory element can also result in chromatin structure changes thus indirectly regulating gene expression. These structural changes may then enhance the probability of binding of other transcriptional activators, as in the case of tyrosine aminotransferase. The hepatic transcription factor HNF3, which has binding sites in albumin enhancers, relieves chromatin repression and thereby activates transcription [Crowe et al. J Biol Chem. 1999 274(35):25113-25120]. This form of regulation may be especially important for gene transfer vectors which, by definition, will not be in their cognate chromosomal environment. The inclusion of Locus Control Regions (LCRs) or matrix attachment regions (MARs) in pDNA expression vectors may also enhance expression of transgenes in specific tissues.
A large number of transcription factors are involved in regulating expression of genes in vivo. For instance, the following 21 liver related regulatory elements (and their respective by length) have been recognized to date: ANF (10), ANF4 (12), AT-rich (13), C/EBP (10), connexin 32 gene B2 element (24), COUP/EAR (16), DBP (13), EP (15), HLF (10), HNF1 (14), HNF3 (9), HNF4 (12), insulin response element (13), metal response element (15), methylated DNA-binding protein site (13), peroxisome proliferator response element (13), promoter linked coupling element (12), Slp gene promoter element (8), VBP (10), xenobiotic response element (10), and Y box (12) (Transcription Element Search Software, Transfac database).